Project Summary Asparaginase (ASNase) is one of the main drugs used for the treatment of pediatric acute lymphoblastic leukemia (ALL). The most common adverse reaction of ASNase is the development of an immune response that can compromise drug exposure and lead to worse treatment outcome. Therefore, therapeutic strategies that prevent or overcome the immune response to ASNase and retain sufficient drug exposure are urgently needed. PEGylated ASNase (PEG-ASNase) is the first-line agent used for ALL therapy, and it remains prone to immunotoxicity during treatment. Our preliminary results showed that: 1) ASNase-induced immune responses can decrease drug exposure through direct drug neutralization and by accelerating the pharmacokinetic clearance of ASNase; 2) therapeutic drug levels can be attained after sensitization to PEG-ASNase but may require drug doses higher than in nave patients to achieve similar therapeutic effects; 3) there is a higher risk of ASNase hypersensitivity among carriers of the nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 (NFATC2) rs6021191 variant, which leads to higher expression of this transcription factor involved in the regulation of immune responses; 4) both IgG and IgE antigen-specific antibodies are involved in the mechanism of ASNase hypersensitivity, which will be used to facilitate the development of a cell-based approach to monitor PEG-ASNase immunogenicity; and 5) we have established two mouse models of transplantable B cell ALL that can be used to study drug efficacy and toxicity. Based on these observations, we hypothesize that (1) pretreatment strategies that can overcome immune responses to PEG-ASNase by blocking the mediators of hypersensitivity and restoring drug concentrations can maintain the antileukemic properties of PEG-ASNase. (2) Genetic and pharmacological inhibition of NFATC2 can prevent or attenuate sensitization to PEG-ASNase. (3) IgG and/or IgE-mediated binding of peripheral blood cells (PBCs) to PEG-ASNase can be used as a biomarker of immunotoxicity. We propose three specific aims to test our hypotheses: 1) to determine whether combining pretreatment drugs that block the mediators of hypersensitivity and PEG-ASNase dose adjustments can overcome immune responses and retain antileukemic efficacy using two murine models of PEG-ASNase hypersensitivity and B cell ALL; 2) to determine whether inhibition of the NFAT pathway can prevent sensitization to PEG-ASNase and maintain antileukemic efficacy; and 3) to determine if sensitized PBCs can be identified through their binding to fluorescently labeled PEG-ASNase ex vivo using flow cytometry. The proposed work will identify strategies to overcome, prevent, and monitor the immune response to PEG- ASNase. The three aims are not interdependent but are logically related with a singular focus on improving PEG- ASNase therapy. The long-term goal of this project is to improve pediatric ALL survival by overcoming or preventing the drug-induced immune response and ensuring that patients receive adequate PEG-ASNase exposure during ALL treatment.